Design and synthesis of tag-free photoprobes for the identification of the molecular target for CCG-1423, a novel inhibitor of the Rho/MKL1/SRF signaling pathway

Jessica L Bell; Andrew J Haak; Susan M Wade; Yihan Sun; Richard R Neubig; Scott D Larsen

doi:10.3762/bjoc.9.111

Design and synthesis of tag-free photoprobes for the identification of the molecular target for CCG-1423, a novel inhibitor of the Rho/MKL1/SRF signaling pathway

Beilstein J Org Chem. 2013 May 21:9:966-73. doi: 10.3762/bjoc.9.111. Print 2013.

Authors

Jessica L Bell¹, Andrew J Haak, Susan M Wade, Yihan Sun, Richard R Neubig, Scott D Larsen

Affiliation

¹ Vahlteich Medicinal Chemistry Core, Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA.

Abstract

CCG-1423 and related analogues represent a new class of inhibitors of Rho/MKL1/SRF-mediated gene transcription, a pathway that has been implicated in both cancer and fibrosis. The molecular target for these compounds is unknown. To facilitate its identification, a series of tag-free photoaffinity probes was designed and synthesized, each one containing a photoactivatable group and an acetylenic end group for subsequent attachment to a fluorescent tag using click chemistry. All were confirmed to maintain biological activity in a cell-based assay for inhibition of SRE-Luc expression. The functional activity of the most potent probe 24 was further confirmed in an assay for PC-3 cell migration. Photolysis of 24 in intact PC-3 cells followed by cell lysis, click ligation of a fluorescent dye, and gel electrophoresis revealed specific labeling of a single 24 kDa band that could be blocked with an active competitor. Future work will focus on identifying the labeled protein(s).

Keywords: CCG-1423; Rho pathway inhibitor; click ligation; photoaffinity labeling; tag-free photoprobe; target identification.